Soft meningeal cell separation method
Through multi-step processing, optimized leponema cell isolation methods, the problems of impurity removal and cell activity maintenance are solved, and the acquisition and standardized operation of high-purity cells are achieved, which meets the needs of different sample sizes and supports neuroscience research, drug development and clinical treatment.
Patent Information
- Application Number
- CN202510720175.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-30
- Publication Date
- 2025-08-15
AI Technical Summary
The prior art is difficult to accurately remove impurity cells in leptomy tissue, unable to maintain cell activity, lack of standardized operating procedures, resulting in poor repetition of experimental results, and low efficiency and vulnerability to cells when processing small sample sizes.
Optimize digestion and centrifugation conditions to ensure cell purity and activity through multi-step treatment including dust-free paper rolling, cold KRBS BUFFER rinsing, enzyme digestion, centrifugation and Percoll density gradient centrifugation.
Obtain high-purity and high-active leponema cells, reduce experimental interference, and are easy to operate and repeatable, adapt to different sample size needs, and meet the needs of neuroscience research, drug research and development and clinical treatment.
Smart Images
Figure CN120485117A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of cell biology, and in particular to a method for isolating pia mater cells. Background Art
[0002] The study of pia mater cells is of vital importance in the fields of neuroscience research, drug development, and clinical treatment. As a layer of connective tissue membrane that is closely attached to the surface of the brain, the pia mater not only provides physical protection for brain tissue, but also participates in the formation of the blood-brain barrier, playing a key role in maintaining the stability of the internal environment of the central nervous system. Dysfunction of pia mater cells is closely related to the occurrence and development of various neurological diseases, such as neuroinflammation, neurodegenerative diseases, and brain tumors. Therefore, obtaining high-purity and high-activity pia mater cells is crucial for in-depth exploration of their physiological functions, pathological mechanisms, and the development of targeted treatment strategies.
[0003] However, the isolation and extraction of pial cells currently faces many challenges. Traditional cell isolation methods have obvious limitations:
[0004] On the one hand, it is difficult to accurately remove the cerebral white matter, vascular blood cells, and other connective tissue components that are mixed in with the pia mater tissue. The presence of these impurity cells can interfere with the accurate study of pia mater cells in subsequent experiments, leading to biased experimental results and failing to truly reflect the biological characteristics and functions of pia mater cells. For example, when studying the gene expression profile of pia mater cells, the gene expression of impurity cells can mask the characteristic expression of pia mater cells, affecting the accuracy of research conclusions.
[0005] On the other hand, existing separation technologies often fail to maintain the activity of pia mater cells well. During the separation process, excessive mechanical damage, inappropriate enzymatic digestion conditions, and prolonged operation may lead to reduced cell activity or even death. Cells with low activity are difficult to grow and proliferate normally during culture, and cannot meet the needs of long-term experiments, which limits in-depth research on the function of pia mater cells. Moreover, due to the large differences in isolation methods among different laboratories and the lack of standardized operating procedures, the reproducibility of experimental results is poor, making it difficult to effectively compare and verify different studies.
[0006] In addition, in actual research, the amount of pia mater tissue samples obtained is usually small. When dealing with small sample sizes, existing separation methods often cannot efficiently separate a sufficient number of pia mater cells, or cause excessive damage to the cells during the separation process, further exacerbating the difficulty of pia mater cell research.
[0007] Therefore, we proposed a method for isolating pial cells to address the above-mentioned problems.
[0008] The above information disclosed in this background technology is only for enhancing understanding of the background technology of the present invention and therefore it may contain information that does not constitute the prior art that is already known to a person of ordinary skill in the art. Summary of the Invention
[0009] The present invention aims to address one of the issues identified in the aforementioned background art regarding the current pial cell isolation and extraction process: difficulty in accurately removing impurity cells, inability to maintain cell viability, lack of standardized operating procedures resulting in poor reproducibility of experimental results, and low efficiency and susceptibility to cell damage when processing small sample sizes. The present invention provides a pial cell isolation method that, by obtaining high-purity and high-activity pial cells, addresses the research needs of pial cells in fields such as neuroscience research, drug development, and clinical treatment.
[0010] To achieve the above object, the present invention provides the following technical solutions:
[0011] A method for isolating pia mater cells comprises the following steps:
[0012] S1. Obtain pial tissue and roll it on cleanroom paper to remove cerebral white matter and large blood vessels.
[0013] S2. Rinse the tissue in a cold KRBS buffered cell culture-grade dish to remove residual white matter and blood vessels, retaining the cortex.
[0014] S3. Cut the tissue into 1mm pieces 3 Size, add digestion solution containing type P collagenase and DNase I, digest at 37℃ for 1-1.5 hours;
[0015] S4. After digestion, centrifuge and resuspend the pellet in 20% BSA. Perform gradient centrifugation to remove impurities and retain the cell pellet at the bottom.
[0016] S5. Add trypsin digestion solution and digest for 10 minutes. After serum-terminated digestion, collect cells by centrifugation.
[0017] S6. Depending on the sample size, choose direct culture or Percoll density gradient centrifugation for purification.
[0018] S7. Wash the purified pial cells and freeze or culture them in an incubator.
[0019] Preferably, in step three, the concentration of collagenase is 2 mg / ml, and the concentration of DNase I is 30 U / ml.
[0020] Preferably, in step 4, the conditions for gradient centrifugation are 1000 g, 20 minutes, and 4°C.
[0021] Preferably, in step six, the conditions for Percoll density gradient centrifugation are 800 g, 10 minutes, and 4° C., and the gradient solution consists of 11%, 20%, 23%, and 25% Percoll solutions.
[0022] Preferably, in step six, the pia mater cells are located at the 23% and 25% interface, and the cloudy cell layer at the 23%-25% interface is aspirated with a tube and transferred to a new centrifuge tube.
[0023] Preferably, in step seven, 3 ml of HBSS containing EGTA, BSA and glucose is added for washing twice, and the mixture is centrifuged at 1000 rpm for 5 minutes at room temperature.
[0024] Application of leptomeningeal cells in neuroscience research, drug screening or clinical treatment.
[0025] Compared with the prior art, the present invention has the following beneficial effects:
[0026] In the present invention, impurities such as brain white matter and vascular blood cells are effectively removed through multi-step processing, and high-purity pia mater cells can be obtained, reducing interference with subsequent experiments. The operation is simple and standardized, the parameters of each step are clear, and the repeatability is strong, which is convenient for promotion in different laboratories. The separation method of the present invention focuses on cell protection throughout the process, optimizes digestion and centrifugation conditions, and the cells can grow and proliferate normally in subsequent culture. If the sample amount is small, some steps can be omitted and used directly for detection or culture. If the sample is sufficient, the purity can be improved according to the complete process. It provides high-quality cell samples and technical support for neuroscience research, drug screening, and clinical treatment.
[0027] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present invention will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 This is a flow chart of pia mater separation according to the present invention;
[0029] Figure 2 This is one of the operational flow charts of the method for isolating pial cells of the present invention;
[0030] Figure 3 This is the second operational flow chart of the method for isolating pial cells of the present invention;
[0031] Figure 4 This is the third operational flow chart of the method for isolating pia mater cells of the present invention. DETAILED DESCRIPTION
[0032] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.
[0033] Example 1
[0034] See also Figures 1-4 , a method for isolating pial cells, comprising the following steps:
[0035] 1. Tissue acquisition and pretreatment:
[0036] Clinical discarded pial tissue samples were obtained through the hospital's ethical approval and compliance procedures.
[0037] Place the meningeal tissue containing a small amount of white matter on sterile dust-free paper and gently roll it 5-8 times to remove visible white matter and large blood vessels, and try to remove non-pial membrane parts such as brain white matter and vascular blood cells.
[0038] The cells were transferred to a 60 mm culture dish containing pre-cooled KRBS buffer (cell culture grade), and the residual white matter and blood vessels were carefully removed using 25G microtweezers, leaving the translucent cortical layer.
[0039] 2. Enzyme digestion treatment:
[0040] Rinse the tissue three times with ice-cold KRBS buffer, gently shaking the dish for 15 seconds each time.
[0041] Add 1 ml of DMEM / F12 basal culture medium and use iris scissors to mince the tissue into pieces of about 1 mm. 3 Small uniform pieces, about 50-80 pieces.
[0042] Add 2 ml of digestion solution containing 2 mg / ml type P collagenase (Sigma-C9891) and 30 U / ml DNaseI (Sigma-DN25) and mix gently by pipetting.
[0043] Digestion was carried out in a 37°C water bath at 100 rpm for 60 minutes, with inversion mixing every 15 minutes.
[0044] 3. Preliminary separation and purification:
[0045] The digested product was transferred to a 15 ml centrifuge tube and centrifuged at 1000 rpm at 4°C for 5 minutes.
[0046] The supernatant was discarded, 5 ml of 20% BSA (Sigma-A7906) solution was added, and the precipitate was resuspended by pipetting.
[0047] Centrifuge at 4°C, 1000 g for 20 minutes, carefully aspirate the middle fat and connective tissue layer, and retain the cell pellet at the bottom.
[0048] 4. Secondary enzymatic hydrolysis and single cell suspension preparation:
[0049] Add 3 ml of 2.5% trypsin digestion solution (Gibco-25200) to the precipitate and incubate at 37°C for 10 minutes, gently tapping the bottom of the tube every 2 minutes.
[0050] Add 3 ml of DMEM culture medium containing 10% FBS to terminate the digestion, and pipette 10 times.
[0051] Centrifuge at 1000 rpm at 4°C for 8 minutes, discard the supernatant, and resuspend the pellet in 2 ml of HBSS buffer.
[0052] 5.Percoll density gradient centrifugation purification:
[0053] Take a 15ml centrifuge tube and add 3ml and 25% Percoll solution in sequence.
[0054] Alternatively, add the following solutions to 3 ml of cell suspension (layered on 25% Percoll):
[0055] Slowly add 3 ml of 23% Percoll solution, 2 ml of 20% Percoll solution, and 2 ml of 11% Percoll solution along the tube wall, and centrifuge at 800 g for 10 minutes at 4°C.
[0056] When the amount of tissue obtained is small, Percoll separation is not required. The digestion can be terminated directly and the cell suspension can be used for flow cytometry detection or subsequent culture.
[0057] 6. Cell Collection and Processing:
[0058] After centrifugation, most pia mater cells are located at the 23% and 25% interface, and some are located at the 25% interface. Discard the solution containing anything other than pia mater, and use a Pasteur pipette to aspirate the cloudy cell layer at the 23%-25% interface and transfer it to a new centrifuge tube.
[0059] The cells were washed twice by adding 3 ml of HBSS containing EGTA, BSA and glucose, and centrifuged at 1000 rpm for 5 minutes at room temperature.
[0060] After cell counting, the cells were divided into two parts: frozen and placed in a 37°C, 5% CO2 incubator for static culture to treat the final pial cells.
[0061] The difference between the second embodiment and the first embodiment is that:
[0062] During enzymatic digestion, rinse the tissue three times with ice-cold KRBS buffer, gently shaking the dish for 15 seconds each time.
[0063] Add 1 ml of DMEM / F12 basal culture medium and use iris scissors to mince the tissue into pieces of about 1 mm. 3 small uniform pieces.
[0064] Add 2 ml of digestion solution containing 2 mg / ml type P collagenase (Sigma-C9891) and 30 U / ml DNaseI (Sigma-DN25) and mix gently by pipetting.
[0065] Digestion was carried out in a 37°C water bath at 100 rpm for 1.5 h, with inversion mixing every 15 minutes.
[0066] For secondary enzymatic hydrolysis and single-cell suspension preparation, 3 ml of 2.5% trypsin digestion solution (Gibco-25200) was added to the pellet and incubated at 37°C for 20 minutes, gently tapping the bottom of the tube every 2 minutes.
[0067] Add 3 ml of DMEM culture medium containing 10% FBS to terminate the digestion, and pipette 10 times.
[0068] Centrifuge at 1000 rpm at 4°C for 8 minutes, discard the supernatant, and resuspend the pellet in 2 ml of HBSS buffer.
[0069] The above are all preferred embodiments of the present application, and are not intended to limit the scope of protection of the present application. Therefore, any equivalent changes made based on the structure, shape, and principle of the present application should be included in the scope of protection of the present application.
[0070] In summary, the present invention effectively removes non-pial tissue components, such as cerebral white matter and vascular blood cells, through a multi-step process. In the initial processing stage, visible impurities are removed to the greatest extent possible by rolling the meningeal tissue on dust-free paper and carefully manipulating it with fine dissecting forceps. Subsequent steps such as enzymatic digestion, centrifugation, and density gradient centrifugation further screen out pia mater cells. The pia mater cells isolated using this method are of high purity, reducing the interference of impure cells on subsequent experimental results and providing cell samples for in-depth research into the biological properties, functions, and related disease mechanisms of pia mater cells.
[0071] The present invention takes into account the protection of pia mater cell activity throughout the entire isolation and extraction process. Using cold KRBSBUFFER for initial tissue treatment and rinsing can reduce the cell metabolic rate and minimize cell damage. The time and temperature of enzymatic digestion are optimized to avoid irreversible damage to cells caused by excessive digestion. During centrifugation, the appropriate rotation speed and time are used to reduce the impact of mechanical forces on the cells. The isolated pia mater cells can adhere normally, grow, and proliferate in subsequent culture, maintaining good cell activity and meeting the needs of long-term culture and various experiments.
[0072] The present invention is highly adaptable to varying sample sizes. When obtaining a small amount of pial tissue, the Percoll density gradient centrifugation step can be omitted, and the cell suspension can be used directly for flow cytometry or subsequent culture. This avoids the possibility of experimental inconvenience due to insufficient sample size and improves sample utilization. For sufficient sample sizes, the complete process can be followed to further improve cell purity.
[0073] The isolated and extracted pia mater cells provide a research foundation for multiple fields, including neuroscience research, drug screening, and clinical treatment. In neuroscience research, high-purity, well-active pia mater cells can help to further explore the development of the nervous system, the mechanism of neural signal transduction, and the pathogenesis of neurological diseases. In drug screening, these cells can be used to construct in vitro models to more accurately assess the effects and safety of drugs on pia mater cells, accelerating the development of new drugs. In the field of clinical treatment, pia mater cells may serve as a potential cell source for cell therapy, providing new ideas and methods for the treatment of related diseases.
[0074] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.
[0075] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.
Claims
1. A method for isolating pia mater cells, characterized in that: The following steps are involved: S1. Obtain pial tissue and roll it on cleanroom paper to remove cerebral white matter and large blood vessels. S2. Rinse the tissue in a cold KRBS buffered cell culture-grade dish to remove residual white matter and blood vessels, retaining the cortex. S3. Cut the tissue into 1mm pieces 3 Size, add digestion solution containing type P collagenase and DNase I, digest at 37℃ for 1-1.5 hours; S4. After digestion, centrifuge and resuspend the pellet in 20% BSA. Perform gradient centrifugation to remove impurities and retain the cell pellet at the bottom. S5. Add trypsin digestion solution and digest for 10 minutes. After serum-terminated digestion, collect cells by centrifugation. S6. Depending on the sample size, choose direct culture or Percoll density gradient centrifugation for purification. S7. Wash the purified pial cells and freeze or culture them in an incubator.
2. A method for isolating pia mater cells according to claim 1, characterized in that: In step 3, the concentration of collagenase was 2 mg / ml, and the concentration of DNase I was 30 U / ml.
3. A method for isolating pia mater cells according to claim 1, characterized in that: In step 4, the conditions for gradient centrifugation were 1000 g, 20 min, and 4°C.
4. A method for isolating pia mater cells according to claim 1, characterized in that: In step six, the conditions for Percoll density gradient centrifugation are 800 g, 10 minutes, and 4° C., and the gradient solution consists of 11%, 20%, 23%, and 25% Percoll solutions.
5. A method for isolating pia mater cells according to claim 1, characterized in that: In step 6, the pial cells are located at the 23% and 25% interface. Use a pipette to aspirate the cloudy cell layer at the 23%-25% interface and transfer it to a new centrifuge tube.
6. A method for isolating pia mater cells according to claim 1, characterized in that: In step seven, the cells were washed twice by adding 3 ml of HBSS containing EGTA, BSA, and glucose, and centrifuged at 1000 rpm for 5 minutes at room temperature.
7. Use of the leptomeningeal cell according to any one of claims 1 to 6 in neuroscience research, drug screening or clinical treatment.